Pectin is a complex polysaccharide associated with plant cell walls. It consists of an alpha 1-4 linked polygalacturonic acid backbone intervened by rhamnose residues and modified with neutral sugar side chains and non-sugar components such as acetyl, methyl, and ferulic acid groups.
The neutral sugar side chains, which include arabinan and arabinogalactans, are attached to the rhamnose residues in the backbone. The rhamnose residues tend to cluster together on the backbone. So, with the side chains attached this region is referred to as the hairy region and the rest of the backbone is hence named the smooth region.
Pectins are traditionally used as food additives. However, their use has extended into pharmaceutical areas as well. Pectins have long been used as an anti-diarrhea agent and can improve intestinal functions. The anti-diarrhea effect is thought to be in part due to pectin's anti-microbial activity.
Pectins are also effective against gastrointestinal ulcers and enterocolitis. Pectins also influence cell proliferation in the intestines. They also have blood cholesterol lowering effect and exhibit inhibition of atherosclerosis. This effect is the result of interactions between pectins and bile salts. Pectins have also been shown to affect the fibrin network in hypercholesterolaemic individuals.
The ability to interact with many divalent metal ions renders pectins a strong detoxifying agent.
The resistance of pectin to degradation in the upper GI tract and its complete dissolution in the colon makes pectin very suited for colon-specific delivery. Coacervation with gelatin permits the formation of microglobules suitable for controlled-release products. Further, pectin is used in tablet formulations.
According to Kertesz, Z. I: The Pectic Substances, Interscience Publishers, Inc, New York, 1951, pectic materials occur in all plant tissues. However, of industrial importance are particularly apples, beets, flax, grapefruit, lemons, limes, oranges, potatoes, and sunflower. Lately, also the pectin in Aloe vera has shown industrial utility.
Historically, pectin has mainly been used as a gelling agent for jam or similar, fruit-containing, or fruit-flavored, sugar-rich systems. Examples are traditional jams, jams with reduced sugar content, clear jellies, fruit-flavored confectionery gels, non-fruit-flavored confectionery gels, heat-reversible glazings for the bakery industry, heat-resistant jams for the bakery industry, ripples for use in ice cream, and fruit preparations for yoghurt.
A substantial portion of pectin is today used for stabilization of low-pH milk drinks, including fermented drinks and mixtures of fruit juice and milk.
Lately, pectins have been found to be effective for the treatment of heartburn caused by esophagus acid reflux.
The galacturonic acid residues in pectin are partly esterified and present as the methyl ester. The degree of esterification is defined as the percentage of carboxyl groups esterified. Pectin with a degree of esterification (“DE”) above 50% is named high methyl ester (“HM”) pectin or high ester pectin and one with a DE lower than 50% is referred to as low methyl ester (“LM”) pectin or low ester pectin. Most pectin found in fruits and vegetables are HM pectins. Acetate ester groups may further occur at carbon-2 or -3 of the galacturonic acid residues. The degree of acetate esterification (“DAc”) is defined as the percentage of galacturonic acid residues containing an acetate ester group. Most native pectins have a low DAc, one exception being sugar beet pectin.
Pectins are soluble in water and insoluble in most organic solvents. Pectins with a very low level of methyl-esterification and pectic acids are for practical purposes only soluble in the form of a potassium or sodium salt.
Pectins are most stable at pH 3-4. Below pH 3, methoxyl and acetyl groups and neutral sugar side chains are removed. At elevated temperatures, these reactions are accelerated and cleavage of glycosidic bonds in the galacturonan backbone occurs. Under neutral and alkaline conditions, methyl ester groups are saponified and the polygalacturonan backbone breaks through beta-elimination-cleavage of glycosidic bonds at the non-reducing ends of methoxylated galacturonic acid residues. These reactions also proceed faster with increasing temperature. Pectic acids and LM pectins are resistant to neutral and alkaline conditions since there are no or only limited numbers of methyl ester groups.
There are many biocatalysts that can specifically modify and degrade pectin molecules. These biocatalysts include endo- and exo-polygalacturonase, pectate lyase, pectin methylesterase, pectin acetylesterase, and rhamnogalacturonase. According to Rastall, R.: LFRA Ingredients Handbook, Leatherhead Food RA, March 1999, Pectinases from Aspergillus spp include:
pHBiocatalystEC no.SubstrateActionopt.Temp. opt.Pectin lyase4.2.2.10High ester pectinEndo a-1,4 b5-5.550eliminationPectin methyl3.1.1.11High ester pectinRandom de-4-4.555esteraseesterificationEndo-3.2.1.15Low ester pectinEndo a-1,44-4.545-50polygalacturonasedepolymerizationExo-3.2.1.67Low ester pectinExo a-1,4555polygalacturonasehydrolysisArabinogalactanase3.2.1.90Pectin hairyEndo b-1,3450regions1,6hydrolysisMannanase3.2.1.78Pectin hairyEndo b-1,43-6  80regionshydrolysis
Suppliers include Gist-Brocades, Novo Nordisk A/S, Röhm, Shin Nihon, and Solvay.
Both HM and LM pectins can form gels, but by totally different, mechanisms. HM pectin form gels in the presence of high concentrations of co-solutes, such as sugar at low pH. LM pectins form thermoreversible gels in the presence of calcium. In addition, the sugar beet pectin can form gels through cross-linking of the ferulated groups as taught by Rombouts (U.S. Pat. No. 4,672,034).
Pectins have a favorable regulatory status as a food additive. They are classified as Generally Recognized As Safe (“GRAS”) in the United States and Acceptable Daily Intake (“ADI”) in Europe.
In U.S. Pat. No. 2,480,710, Bryant discloses a process for making amidated pectin. A slow setting pectin type with 7.5% to 10% methoxyl content is suspended in 65% IPA with 10% ammonium hydroxide (28% ammonia), and the mix is stirred for two hours at 25° C. After draining of ammoniacal alcohol, fresh 65% IPA is added. Concentrated HCl is then added to an acidity, which results in a pH of a 1% dispersion of the amidated pectin of 3-4. pH higher than 5 and below 3 is likely to cause further undesirable modification during drying. The suspension is filtered and rinsed with first 65% IPA and then 90% IPA and then dried. The conversion followsRCOOCH3+NH4OH═RCONH2+CH3OH+H2O
The desired amount of ammonia ranges from 2 to 16 times the amount theoretically required to de-esterify completely the original starting material.
Instead of using an isolated pectin, wet pectin precipitate can be used.
In U.S. Pat. No. 2,478,170, William et al. disclose that low ester pectin can be prepared by the partial de-esterification of either pectin or protopectin. William et al use a pectin starting material, which is unmodified and which displays a degree of esterification of about 80%. Acid, alkali or biocatalyst catalyzes this de-esterification. In the disclosure, alkali is used as the de-esterifying agent to reach a degree of esterification from 20% to 30%.
In U.S. Pat. No. 2,448,818, McCready et al. disclose the use of citrus pectinesterase to transform in situ pectin into low ester pectin having a methoxyl content of 6%, corresponding to a degree of esterification of about 37%. The biocatalyst utilized is naturally occurring in citrus fruit, and such plant esterases are known to result in a block wise de-esterification.
In U.S. Pat. No. 3,622,559, Wiles et al. makes a pectin starting material through acid extraction at around 70° C. for 1-2 hours. This leads to a pectin having a molecular weight (Mw) of 180,000 to 200,000 Daltons and a methoxyl content of 10% (DE=61%) to 10.5% (DE=64%). This pectin may then be further de-esterified by treating the pectin with acid or an ammonia-alcohol mixture. This process leads to low ester pectin having molecular weight (Mw) of 120,000 to 200,000 Daltons.
In U.S. Pat. No. 4,065,614, Nelson discloses an amidated pectin having a degree of amidation of at least 27%. The starting pectin material must have a degree of esterification over 60%, and amidation is conducted using a mixture of liquid ammonia in a suitable organic solvent such as isopropanol. The reaction is carried out at a temperature within the range −15-+15° C. until the degree of amidation has reached at least 27%. The pectin starting material is disclosed as any of the commercially available high ester pectins.
In DK 157,616 B, Buhl et al. disclose an amidated pectin having a degree of amidation of 5-25%, a degree of esterification of at least 50% and an average molecular weight of 15,000-75,000 Daltons. The pectin starting material is made according to a conventional acid extraction, and amidation is performed in a mixture of liquid ammonia and isopropanol at 0° C.
In WO 98/58968, Larsen et al. disclose a process for making selected fractions of high ester pectin. The process involves two or more extraction steps characterized in that each proceeding step involves extraction of the pectin from the preceding step at lower pH. The high ester pectin fractions are also useful as starting materials for making de-esterified pectin. The pectin starting material for de-esterification has a DE above 50%, and is treated with a de-esterifying agent to obtain a pectin fraction having a degree of esterification, which is reduced by at least 5% relative to that of the high ester pectin fraction and a degree of amidation in the range 0-25%. De-esterifying agents are acids, alkalis or combinations thereof.
In WO 00/08952, Madsen et al. disclose a pectin composition providing no substantial gelation. It is disclosed that the degree of esterification and/or degree of block structure may be modified using biocatalysts obtained from plants or bacteria.
In WO 00/15830, Christensen et al. disclose a process for treating pectin with a pectin methyl esterase. The pectin methyl esterase is not derived from plant, but has at least one plant pectin methyl esterase property. That property is the block-wise de-esterification of pectin, and the treated pectin is disclosed to contain at least 70% ester groups.
In U.S. Pat. No. 6,069,000, Andersen et al. disclose a biocatalyst with pectin esterase activity. The inventors believe that the improved performance of the pectin esterase of the invention is due to the mode of action of the biocatalyst of the invention preferably providing a block-wise distribution of the acid groups in the pectin.
In U.S. Pat. No. 5,707,847, Christgau et al. have disclosed a biocatalyst exhibiting pectin methyl esterase activity. The biocatalyst can be used for demethylation of pectin, e.g. from citrus, apple, sunflower and/or sugar beet. The biocatalyst can be used to produce low methylated pectin from high methylated pectin.
In this reference, microbial pectin esterase is comparable in its effect to acid and alkali, i.e. de-esterification is random in nature.
In the conventional processes of de-esterifying pectin, chemical processes involve the use of acid or alkali treatment of high ester pectin. These processes are known to result in depolymerization of the high ester pectin during de-esterification, and consequently, chemical de-esterification typically must take place at low temperatures. This on the other hand makes the de-esterification process slow and in addition, investments in cooling equipment make such de-esterified pectins more costly to produce. Further, since the chemical processes are known to remove ester groups in a random fashion, the resulting de-esterified pectins are less blocky with respect to carboxylic acid groups.
Enzymatic processes are known to de-esterify high ester pectin in either a random fashion or in a block wise fashion. Further, enzymatic de-esterification is known to reduce or to eliminate depolymerization of the high ester pectin during de-esterification.
However, enzymatic de-esterification suffers from the fact that the degree of esterification cannot be brought below about 25% as taught by Smythe (U.S. Pat. No. 2,599,531). Additionally, no enzymatic process has been disclosed, which will result in amidated pectins.
Thus, there remains a problem that conventionally-made amidated low ester pectins still suffer from glycosidic breakdown and aggregation during amidation. This leads to amidated pectins with a changed conformation due to aggregation.
The existence of pectin aggregates in solution has been reported in the literature (Sorochan et al. 1971. Carbohydr. Res. vol. 20, pp. 243-248; Jordan, R. C. and Brant, D. A. 1978. Biopolymers. vol. 17, pp. 2885-2895; Davis et al. 1980. Int. J. Biol. Macromol. vol. 2, pp. 330-332). Pectin aggregates form in solution under non-gelling conditions and could be considered precursors to gelation U.S. Pat. No. 6,143,337 to Fishman, et al. This aggregation is speculated to cause the amidated pectin to become less soluble, which leads to a lower gelling power than theoretically possible. Additionally, non-amidated low ester pectins having a degree of esterification below 25% have heretofore been made by either acid or alkaline de-esterification, and for these low ester pectins the same problem exists, namely that acid or alkali hydrolysis reduce the molecular weight of the pectin and consequently produce a low ester pectin with a lower gelling power than theoretically possible.
Consequently, there is a need for a process, which offers                A method for reducing the degree of esterification to below 25% with less breakdown of the glycosidic links in the pectin backbone.        Non-amidated low ester pectins with a degree of esterification below 25% having higher molecular weight and a non-aggregated or less aggregated conformation to provide for an increase in gelling power.        A method for making amidated pectin with higher molecular weight and a less aggregated conformation.        Amidated low ester pectins of higher molecular weight and a non-aggregated or less aggregated conformation to provide for an increase in gelling power.        